Manufacture of isoflavones

ABSTRACT

A process for manufacturing a hydroxylated isoflavone of the formula (I) given in the description comprises re-acting an appropriately substituted 2-hydroxydeoxybenzoin of the formula (II), also given in the description, with a formic acid anhydride of the formula HCOOCOR3, wherein R 3  signifies C 2-20 -alkyl or various other groups as given in the description, in presence of a base or in a solvent which acts as a base, and if necessary promoting the ensuing hydrolysis of the so-produced acylated form of the hydroxylated isoflavone of the formula I by acidification. Of particular interest as products of this process are the 5,7dihydroxyisoflavones, e.g. genistein (5,7,4′-trihydroxyisoflavone). Isoflavones display many useful biochemical effects.

The present invention concerns a process for the manufacture of certainisoflavones from appropriately substituted 2-hydroxyphenyl benzylketones. Of particular interest as products of this process are the5,7-dihydroxyisoflavones, e.g. genistein (5,7,4′-trihydroxyisoflavone).

Isoflavones display many useful biochemical effects. For example, thenaturally occurring and commercially available substance genistein hasbeen claimed to be useful as an anti-inflammatory agent, for preventionand treatment of osteoporosis and heart disease, for prevention ofphotodamage and aging skin and wrinkles, for inhibition of Alzheimersdisease and for treatment of menopausal symptoms, estrogen disorders,cancer, cataracts, cystic fibrosis and migraine. The synthetic andcommercially available isoflavone irpiflavone has been claimed to beuseful for treatment of osteoporosis and estrogen disorders. Amongst thevoluminous literature in this field, M. Messina, Chemistry & Industry1995, 413-415, and T. E. Wiese et al., ibid. 1997, 648-653, presentinteresting reviews on the biological effects and uses of isoflavones,including genistein.

The naturally occurring isoflavones formononetin and biochanin A, whichin contrast to genistein and daidzein do not occur in soy, are ofagricultural interest as promoters for mycorrhizal fungi which benefitplant growth.

Isoflavones are characterized by the following general berizopyranonestructure, the ring numbering also being shown:

Many substances containing the isoflavone structure are found naturally,mainly in the family Leguminosiae; the richest sources are soy, lentils,chickpea, fenugreek, clover, alfalfa and various-types of beans.Genistein, for example, was first isolated in 1899 from broom (Genistatinctoria), and later, in 1931, isolated from Soya hispida [see A. G.Perkins et al., J. Chem. Soc. 75, 830 (1899), and E. Walz, Ann. Chem.489, 118 (1931)]. Its structure was first determined by W. Baker and R.Robinson in 1926 (J. Chem. Soc. 1926, 2713), and the compound latersynthesized (ibid. 1928, 3115).

Most frequently the isoflavones are substituted on the rings to varyingdegrees with hydroxy, alkoxy, prenyl and prenyl-derived groups, and themolecule may constitute part of a more complex ring structure. Naturallyoccurring isoflavones are often substituted at the hydroxy groups withsugars, which on their part are sometimes additionally substituted byester groups. Isoflavone are commonly isolated in nature as mixtureswith closely related substances. For example, isoflavones isolated fromsoybeans include genistein and daidzein which occur co-mixed as freeaglycones (sugar-free forms) as well as their glycosides genistin anddaidzin in soybeans to the extent of about 500-3000 ppm on a dry weightbasis. Direct isolation from biomass containing such mixtures is thuscomplex and often largely economically impracticable.

It is known to experts in the pertinent technical field that othercomponents in natural mixtures may alter the bioavailability and degreeof bioactivity of the active isoflavone principles. In controlledstudies, individual isoflavones from these mixtures also show differingbiological activities, degrees of the desired activities, andside-effects. WO 93/23069, for example, discloses the differingactivities of the isoflavones found in clover. Further recent studiessuggest that isoflavone glycosides are less bioavailable than the freeaglycones. Thus, isolation of isoflavone products from biomass ideallyrequires a hydrolytic step to cleave the glycosides and some type ofreproducible fractionation operation to purify the isoflavones.

Soybeans are one of the few rich nutritional sources of naturalisoflavones. As a high proportion of the isoflavones in soy occursnaturally as water-soluble glycosides, losses to aqueous sidestreams andwastes during protein manufacture occur to varying degrees depending onthe manner of processing [Murphy, J. Agric. Food Chem. 44, 2377-2383(1996)]. Some manufacturers of soy protein seek to retain the desirableisoflavones in soy food products by modifications of normal methods forsoy protein production, for example as described in WO 00/17217, U.S.Pat. No. 6,140,469, and U.S. Pat. No. 5,994,508. Soy proteins can betreated with glycosidases to hydrolyze them, and the resulting aglyconesthen precipitate with the proteins as described in U.S. Pat. No.5,320,949, U.S. Pat. No. 5,352,384, U.S. Pat. No. 5,632,561 and U.S.Pat. No. 5,637,562. Such methods provide food products with varyingamounts of mixed isoflavones. However, apart from tofu and a few otherforms of soy protein, human consumption of soy is not very prevalent inthe Western World.

Isoflavone glycosides can be recovered from soy processing streams byvarious means. For example, U.S. Pat. No. 5,670,632 describes a processfor the isolation of a concentrated mixture of isoflavone glycosides byion-exchange adsorption/desorption. U.S. Pat. No. 5,679,806 describes aprocess for adsorption/desorption with polymeric resins of isoflavoneglycosides from soy molasses or soy extracts. Under conditions ofgradient elution chromatography, the glycosides maybe separatedindividually, isolated and purified. In U.S. Pat. No. 5,702,752 there isdescribed a process of ultrafiltration for the recovery of mixedisoflavone glycosides. U.S. Pat. No. 6,033,714 describes a furtherprocess of ultrafiltration by which genistin, the glycoside ofgenistein, is selectively separated from soy molasses or soy whey. Eachof these processes requires further purification of the glycoside andits hydrolysis to obtain the respective pure isoflavone.

Soy fractions are also used as part of the culture media forfermentations. As described in U.S. Pat. No. 5,554,519, genistein can beisolated as a by-product of the production of erythromycin. However, theproduction capacity is low and care must be taken to remove otherfermentation products from the isoflavones in order to safely use themfor human administration.

Unusually, a high concentration of isoflavones can be found in thebiomass of plants of normal genetic origin. For example, clover containsgenistein, daidzein, formononetin, biochanin and their glycosides in atotal concentrations of 0.5 to 3.5% on a dry weight basis. Due to thehigher content, isolation is more economically feasible. Thus, U.S. Pat.No. 6,146,668 describes solvent extraction of red clover in the presenceof glycoside-hydrolyzing enzymes to obtain mixed isoflavones. These canthen be further separated by extraction and crystallization to obtainindividual isoflavones, such as genistein. The extraction generateslarge amounts of solvent-laden waste biomass. Furthermore, oneconsequence of the purification of genistein is the unavoidableco-production of significant amounts of daidzein which is of lesservalue, as well as bioactive organic wastes.

The above examples serve to illustrate some of the disadvantages ofisolating or otherwise obtaining isoflavones from higher abundancenatural sources. Other useful or potentially useful isoflavones occurnaturally in very low amounts and are thus not practical to isolate. Yetothers are not directly available from natural sources but can besynthesized, albeit inefficiently and uneconomically. Thus there existsa need for practical procedures for efficiently manufacturing largeamounts of pure isoflavones.

A number of chemical syntheses of isoflavones have been developed overmany years, as reported in various reviews in the general and specialistliterature of organic chemistry, such as The Chemistry of FlavonoidCompounds, Geissmann (ed.), Pergamon Press 1962; The Flavonoids,Harborne, Mabry and Mabry (eds), Academic Press 1975; The Flavonoids,Harborne (ed.), Chapman-Hall 1986; and Ellis, General Methods ofPreparing Chromones, Chapter IX in Chromans, Chromenes and Chromanones,Wiley & Sons, 1972. The major synthetic procedures include rearrangementof flavanones and chalcones, condensations of phenols with beta-ketoacids, esters or nitriles, coupling of substituents to preformed flavonerings, and acylations of 2-hydroxyaryl benzyl ketones(“2-hydroxydeoxybenzoins”).

In the earlier years, from about 1928 to about 1952, two syntheticroutes to genistein and related isoflavones were predominantlyavailable. The first route involved the reaction of a2-hydroxydeoxybenzoin with a cinnamic acid ester, e.g. ethyl cinnamate,to afford the corresponding 2-(2-phenylethenyl)-isoflavone, followed byoxidation with potassium permanganate and thermal decarboxylation to theisoflavanone compound: see W. Baker et al., J. Chem. Soc. 1926, 2713;ibid. 1928, 3115; and ibid. 1933, 274. The second route involved thereaction of a 2-hydroxydeoxybenzoin with sodium and ethyl formate toproduce the appropriate 2H-isoflavone compound directly: see H. S. Mahalet al., J. Chem. Soc. 1934, 1769; F. Wessely et al., Chem. Ber. 66, 685(1933); and R. L. Shriner, J. Org. Chem. 10, 288 (1945). Both routes inwhatever variation led to inadequate yields of the desired isoflavonecompound.

In later years, from about 1952, further methods were developed forsynthesizing isoflavone compounds starting from appropriate2-hydroxydeoxybenzoins [see A. Pelter et al., Synthesis 1978, 326 and843, and Y.-C. Chang et al., J. Agric. Food Chem. 42, 1869 (1994)].Three principle methods were employed for cyclizing the2-hydroxydeoxybenzoin to the isoflavone compound: the first utilizedformamide dimethylacetal and a large excess (at least eight equivalents)of the Lewis acid boron trifluoride etherate in dimethyl formamide[method (i)], the second involved the reaction of the2-hydroxydeoxybenzoin with ethyl chlorooxalate in pyridine followed bysaponification with ethanolic potassium hydroxide and thermaldecarboxylation [method (ii)], and in the third method the2-hydroxydeoxybenzoin was reacted with 1,3,5-triazine in the presence ofboron trifluoride etherate and the mixture treated with acetic anhydridein acetic acid [method (iii)]: see specifically J. Chang et al., J.Agric. Food Chem. 42, 1869 (1994) and A. Pelter et al., Synthesis 1978,843; W. Baker et al., J. Chem. Soc. 1953, 1852-1860; and H. C. Iha etal., Angew. Chem. 23, 129 (1981), respectively. The disadvantages ofthese three principle methods include the use of excess amounts of thecorrosive and environmentally unacceptable boron trifluoride etherate orof amine waste products. Moreover, the method (ii) is complicated ininvolving essentially three reaction steps, and requires hightemperatures for the decarboxylation. In all cases only moderate yields,at best, of the isoflavone compound were achieved.

Still further methods include:

-   -   (iv) reaction of an appropriate polyhydroxy substituted        acetophenone, e.g. 2,4,6-trihydroxyacetophenone, of which some        of the hydroxy groups are protected with hydroxy-protected        p-hydroxybenzaldehyde, and subsequent oxidative cyclization with        thallium (III) nitrate [L. Farkas et al., J. Chem. Soc. Perkin        Trans. I, 305 (1974) and H. Sekizaki et al., Chem. Pharm. Bull.        36, 4876 (1988)];    -   (v) reaction of a 2-hydroxydeoxybenzoin with tert.        butoxy-bis(dimethylamino)methane [K. C. Luk et al., J. Nat.        Prod. 46, 852 (1983), P. F. Schuda et al., J. Org. Chem. 52,        1972-1979 (1987) and S. Sepulveda-Boza, Synth. Commun. 31,        1933-1940 (2001)];    -   (vi) reaction of a 2-hydroxydeoxybenzoin with zinc cyanide        followed by cyclization with hydrochloric acid [L. Farkas, Chem.        Ber. 90, 2940 (1957) and L. Farkas et al., ibid. 91, 2858        (1958)];    -   (vii) reaction of a 2-hydroxydeoxybenzoin with an activated        dimethylformamide under various conditions [S. A. Kagal et al.,        Tetrahedron Lett. 1962, 593, A. C. Jain et al., Ind. J. Chem.        25B, 649-651 (1986), V. S. Parmav et al., Synth. Commun. 18,        511-517 (1988), R. J. Bass, J. Chem. Soc. Chem. Commun. 1976,        78, Y. C. Chang et al., J. Agric. Food Chem. 42, 1869 (1994), S.        Sepulveda-Boza, Synth. Commun. 31, 1933-1940 (2001), K. Wähala        et al., J. Chem. Soc. Perkin Trans. I, 1991, 3005 and K. Wähala        et al., Proc. Soc. for Exper. Biol. and Med., 208, 27-32        (1995)]; and    -   (viii) reaction of a 2-hydroxydeoxybenzoin with a trialkyl        orthoformate under basic conditions [L. Parkas et al., J. Chem.        Soc. Perkin Trans. I 1974, 305, U.S. Pat. No. 5,247,102,        Japanese Patent Publication (Kokai) 09/157,268 (1997) and A.        Levai et al., Synth. Commun. 22, 1735-1750 (1992)].

A further approach pursued by various groups of chemists has involvedthe use of the mixed carboxylic acid anhydride formic-acetic anhydrideas a reagent for reacting with a 2-hydroxydeoxybenzoin, with or withoutthe presence of a base, to produce isoflavones such as genistein andbiochanin A. Earlier investigations of this approach are reported by G.I. P. Becket et al. in Tetrahedron Lett. 1976, 719, and J. Chem. Res.Synop. 1978, 47, and by D. F. Liu et al. in J. Heterocyclic Chem. 28,1641-1642 (1991). More recently, various groups around V. G. Pivovarenkohave prepared many isoflavones, including those with either a hydroxy, amethoxy or a substituted phenyl group as the 3-substituent: see forexample J. Heterocyclic Chem. USSR (Engl. translation) 5, 496-501 (1991)[translated from Khim. Get. Soed. 5, 625-631 (1991)], J. HeterocyclicChem. USSR (Engl. translation) 5, 497-502 (1992) [translated from Khim.Get. Soed. 5, 595-600 (1992)] and Soviet Union (SU) Patent 1,333,674. Inthe reported syntheses Pivovarenko et al. used a large excess of thereagent acetic-formic anhydride, indeed as much as a 50 to 70 timesexcess; and likewise a large excess of base, e.g. about six equivalents.Such bases as sodium formate and tertiary amines, e.g. trimethylamineand tribenzylamine, have been employed. Furthermore, lengthy reactiontimes of up to 6 days appeared to have been necessary. Some reactionswere effected in a solvent, others without.

It is known that acetic-formic anhydride decomposes noticeably at O° C.into carbon monoxide and acetic acid, this decomposition becomingcorrespondingly more rapid as the temperature is raised. The reagent canbe handled with appropriate safety precautions on the small scale, butbecomes increasingly more difficult to handle safely on the large scale(see Giumini, Tetrahedron Lett. 1977, 3071, and P. Strazzolini et al.,Tetrahedron 46, 1081-1118 (1990). As a further effect of the tendency ofacetic-formic anhydride to decompose, the liberated acetic acid in thereactions of the anhydride with 2-hydroxydeoxybenzoins leads to theundesirable formation of the corresponding 2-methylisoflavones, whichare difficult to separate from the 2H-isoflavones.

The conclusion from the above reported disadvantages of usingacetic-formic anhydride in processes for producing isoflavones is thatthe reagent is unsuitable for use in such processes on the large,particularly commercial, scale.

Accordingly, there exists a need for a process for manufacturing2H-isoflavones, e.g. genistein, which does not feature theaforementioned disadvantages, or at least avoids then to a significantextent. One approach amongst many others would be to replace the reagentacetic-formic anhydride with a reagent giving rise to the formyl moietybut which is selective in reacting with 2-hydroxydeoxybenzoins to afforda single product of high purity, i.e. in not giving rise to by-productsin significant amounts. However, it cannot be predicted which reagentswould be suitable sources of the formyl moiety, i.e. would meet thesedemands.

It has now been surprisingly found that anhydrides of formic acid andcertain other carboxylic acids, apart from acetic acid, are suitableformylating agents for reacting with appropriate 2-hydroxydeoxybenzoinsto produce 7-hydroxy- or 5,7-dihydroxy-2H-isoflavones.

The present invention provides a process for manufacturing a 7-hydroxyor 5,7-dihydroxy-2H-isoflavone (hereinafter “hydroxylated isoflavone” )of the general formula.

wherein R¹ signifies hydrogen or hydroxy, and

-   -   R² signifies hydroxy or C₁₋₆-alkoxy,        characterized by reacting a 2-hydroxydeoxybenzoin of the general        formula        wherein R¹ and R² have the significances given above,        with a formic acid anhydride of the general formula        wherein R³ signifies C₂₋₂₀-alkyl, C₁₋₆-haloalkyl,        (C₁₋₆-alkoxy)methyl, carboxy-C₂₋₆-alkyl, aryl-C₁₋₆-alkyl, a        group —CH₂—(OCH₂CH₂)_(m)—O—C₁₋₆-alkyl, a group —CH(R⁴)═CR⁵R⁶, a        group —CH═CH—COOH, C₃₋₈-cycloalkyl, aryl, heteroaryl,        di(C₁₋₆-alkyl)aminomethyl, diarylaminomethyl, a group        —(CH₂)_(n)—COOR⁷, a group —(CH₂)_(m)—COOCHO, a group        —CH═CH—COOCHO, C₁₋₆-alkoxy, aryloxy or formyloxy,    -   each of R⁴, R⁵ and R⁶, independently, signifies hydrogen)        C₁₋₆-alkyl or aryl,    -   R⁷ signifies hydrogen, C₁₋₆-alkyl or aryl,    -   m signifies an integer 1 to 4, and    -   n signifies zero or an integer 1 to 8,        in the presence of a base or in a solvent which acts as a base,        and if necessary promoting the ensuing hydrolysis of the        so-produced acylated form of the hydroxylated isoflavone of the        formula I by acidification.

The so-produced hydroxylated isoflavone of the formula I is according tothe significances of R¹ and R² one of the following compounds:

genistein (R¹ and R² both signify hydroxy);

daidzein (R¹ and R² signify hydrogen and hydroxy, respectively);

biochanin A (R¹ and R² signify hydroxy and methoxy, respectively);

formononetin (R¹ and R² signify hydrogen and methoxy, respectively);

isoflavones of the formula I wherein R¹ signifies hydrogen or hydroxyand R² signifies C₂₋₆-alkoxy, i.e. an alkoxy group other than methoxy.

In the above definition of the symbols R², R³, R⁴, R⁵, R⁶ and R⁷, andany references below to alkyl groups, any alkyl (from C₃) can bestraight-chain or branched. This applies equally to “alkyl” (includingany alkyl mentioned below in connection with the substitution of aryl orheteroaryl) as such or to the alkyl part of such groups as“C₁₋₆-haloalkyl”, “(C₁₋₆-alkoxy)methyl”, “aryl-C₁₋₆-alkyl”,“—CH₂—(OCH₂CH₂)_(m)—O—C₁₋₆-alkyl”, “di(C₁₋₆-alkyl)aminomethyl” and“C₁₋₆-alkoxy” (significances of R³). An exception to this principle isthe group “carboxy-C₂₋₆-alkyl”, in which the alkyl moiety is alwaysbranched, as will be explained hereinafter.

Any halogen substituent, e.g. in “C₁₋₆-haloalkyl” or as a possiblesubstituent for an aryl or heteroaryl group, is in each case fluorine,chlorine, bromine or iodine. In. “C₁₋₆-haloalkyl” itself there may-beone or more (same or different) halogen substituents, but the grouppreferably features a single halogen substituent.

In the group “di(C₁₋₆-alkyl)aminomethyl” the two C₁₋₆-alkyl groups maybe identical or different.

The expression “aryl”, as such or as the aryl part of “aryl-C₁₋₆-alkyl”,“diarylaminomethyl” or “aryloxy” (significances of R³), means phenyl,1-naphthyl or 2-naphythyl, or such a group featuring one or moresubstituents. Such substituents are particularly those selected fromC₁₋₄-alkyl, C₁₋₄-alkoxy, halogen, nitro, cyano, di(C₁₋₆-alkyl)amino,phenyl, carboxyl, (C₁₋₆-alkoxy)carbonyl and formyloxycarbonyl, wherebywhen two or more substituents are present these can be the same ordifferent. Examples of substituted phenyl groups are p-tolyl,3-methoxyphenyl, 4-methoxyphenyl, 2,5-dimethoxyphenyl,3,4-dimethoxyphenyl, 2-carboxyphenyl and 2-(formyloxycarbonyl)-phenyl.

The expression “heteroaryl”, also a significance of R³, means a 5- or6-membered heterocyclic group of aromatic character featuring as ringmember(s) one or more heteroatoms selected from oxygen, sulphur andnitrogen. Examples of such heteroaryl groups are 2- or 3-furyl, 2- or3-thienyl and 4-pyridyl. As in the case of the aryl groups, theheteroaryl groups can be unsubstituted or substituted by one or moresubstitutents as indicated hereinabove for the aryl groups featuringsubstituents.

On the face of it the groups carboxy-C₂₋₆-alkyl and —(CH₂)_(n)—COOR⁷, inthe case where R⁷ in the latter group signifies hydrogen, may beconsidered to embrace members common to both. However, the former groupis to be understood in the context of the present invention to excludeall those members of the latter group which contain 2 to 6 carbon atoms.Thus the C₂₋₆-alkyl moiety of “carboxy-C₂₋₆-alkyl” is always branched,and does not include methylene, dimethylene, trimethylene and thefurther polymethylenes embraced by —(CH₂)_(n)—.

The above formulae I, II and III embrace in each case isomeric forms,e.g. optically active or inactive and E/Z-isomers, as the case permits,as well as mixtures thereof, unless expressly stated to the contrary.

Of all the possibilities for the formic acid anhydride of the formulaIII, the preferred ones are propionic formic anhydride (R³ signifiesethyl), isobutyric formic anhydride (R³ signifies isopropyl), carbonicmonoformic anhydride methyl ester (R³ signifies methoxy) and carbonicmonoformic anhydride ethyl ester (R³ signifies ethoxy).

The process in accordance with the present invention is carried out byreacting the 2-hydroxydeoxybenzoin of the formula II with the formicacid anhydride the formula III under essentially basic conditions, i.e.in the presence of a base as the catalyst or in an organic solvent whichacts as a base, whereby in the latter case no extra base needs to beincluded in the reaction medium. If a base is employed rather than anorganic solvent which acts as a base, the process is preferably carriedout additionally in an organic solvent. Moreover, the process is carriedout at temperatures conveniently in the range of about −20° C. to about+80° C., preferably at temperatures from about −5° C. to about +45° C.

Suitable organic solvents are, in general, polar or slightly polaraprotic solvents. Such solvents are, for example, aliphatic and cyclicethers, e.g. diethyl ether, diisopropyl ether, dibutyl ether, tert.butyl methyl ether, diethylene glycol dimethyl ether, tetrahydrofuranand dioxan; lower aliphatic nitriles, e.g. acetonitrile andpropionitrile; lower aliphatic esters, e.g. lower alkyl formates andacetates; dimethylsulphoxide; halogenated, particularly chlorinated,lower aliphatic hydrocarbons, e.g. methylene chloride; aromatichydrocarbons, e.g. benzene, toluene and xylenes; and lower aliphaticketones, e.g. acetone, 2-butanone, diethyl ketone and methyl isobutylketone. Solvents which act as bases, and therefore which can be usedwithout the presence of an added base in the reaction medium, includedi(lower alkyl)formamides, e.g. dimethylformamide, dimethylacetamide,tetramethylurea and N-methyl-pyrrolidone. The preferred solvent is onewhich has a boiling point (at atmospheric pressure) of less than 80° C.and which is miscible with ethanol and preferably also with water. Inthese circumstances the employed such solvent can at the termination ofthe reaction be replaced with ethanol or aqueous ethanol, and thehydroxylated isoflavone product of the formula I can be isolated throughcrystallization induced by the addition of water (in general isoflavonesare very sparingly soluble in water).

As the base for the reaction between the 2-hydroxydeoxybenzoin of theformula II and the formic acid anhydride of the formula III there isconveniently used an alkali metal or alkaline earth metal hydroxide,e.g. lithium, sodium or potassium hydroxide; an alkali metal or alkalineearth metal carbonate or bicarbonate, e.g. lithium, sodium or potassiumcarbonate, calcium or magnesium carbonate, lithium, sodium or potassiumbicarbonate, or calcium or magnesium bicarbonate, as appropriate; analkali metal or alkaline earth metal salt of a carboxylic acid with upto 10 carbon atoms, e.g. sodium formate or potassium propionate; analiphatic or mixed aliphatic/aromatic tertiary amine, e.g.trimethylamine, triethylamine, N-ethyldiisopropylamine,N,N-dimethylethanolamine and esters thereof such as2-(dimethylamino)-ethyl acetate, triethanolamine or N,N-dialkylaniline;a nitrogen-containing heterocyclic base, e.g. optionally alkylsubstituted pyridine, a N-alkyl substituted piperidine, a N-alkylsubstituted morpholine such as N-methyl-morpholine, or imidazole; or asecondary or tertiary phosphate, especially of an alkali metal, e.g.trisodium phosphate or tripotassium phosphate. The preferred type ofbase is an alkali metal or carbonate, bicarbonate or formate, or analiphatic or mixed aliphatic/aromatic tertiary amine.

In general there are conveniently present in the reaction mixture about1.5 to about 6 equivalents of the formic acid anhydride of the formulaIII per equivalent of the 2-hydroxydeoxybenzoin of the formula II,preferably about 2.5 to about 5 equivalents of the formic acid anhydrideper equivalent of the 2-hydroxydeoxybenzoin. The base, when employed, isgenerally present in an amount which is up to about 6 equivalents perequivalent of the 2-hydroxydeoxy-benzoin. The optimal amount dependsvery much on such factors as the nature of the base itself and of theemployed solvent, and can be determined by appropriate investigation ofthe effects of reaction parameter variation on the ease of reaction andthe purity and yield of the produced hydroxylated isoflavone.

Moreover, the reaction is conveniently effected at normal pressure, thepressure in general not being critical. Furthermore, the reactionmixture is suitably agitated, in particular stirred, to promote goodadmixture of the components and ensuing efficient reaction.

In general the course of the reaction can be observed by suchconventional analytical techniques as HPLC, e.g. by monitoring theconsumption of the starting 2-hydroxydeoxybenzoin.

In one particular procedure for the reaction the starting formic acidanhydride of the formula III is conveniently first produced by reactingsodium formate with the appropriate acid chloride of formula R³COCl,conveniently in a solvent which is usable for the subsequent (main)reaction of the formic acid anhydride with the 2-hydroxydeoxybenzoin,e.g. acetone. After completion of the reaction to produce the formicacid anhydride the appropriate amount of base is added, followed by theappropriate amount of the 2-hydroxydeoxybenzoin, after which the mainreaction is effected as described above. As a variation of thisprocedure the base and the 2-hydroxydeoxybenzoin can both be addedtogether to the freshly produced formic acid anhydride in the reactionsolvent. After it has been established that most of the reaction to thedesired, albeit in some cases acylated, product has been completed, afinal reaction period, e.g. at somewhat elevated temperature,particularly in the range from about 40 to about 50° C., generallypromotes the completion (cyclization). Otherwise, in situ methodology(one pot) can be employed.

After completion of the reaction of the formic acid anhydride of theformula III with the 2-hydroxydeoxybenzoin of the formula II the productcan in principle be isolated. However, and as indicated above, thedesired product has in many cases by this stage become acylated at itsphenolic hydroxyl groups (7-OH, and the hydroxyl group(s) signified byR¹ and/or R²). Accordingly, any such acylated hydroxyl groups must firstbe hydrolyzed to the free hydroxyl groups. The hydrolysis of theso-produced acylated form of the desired hydroxylated isoflavone isconveniently effected by addition of aqueous mineral acid, e.g.sulphuric acid or hydrochloric acid, and heating the acidified mixture.In the case of sulphuric acid its concentration is conveniently 5 to50%, e.g. 10%. However, even concentrated sulphuric acid, e.g. sulphuricacid at a concentration approaching 100%, may be used if the reactionmixture is diluted with water prior tov the acid addition. Ifhydrochloric acid is used for the acidification to promote thehydrolysis, said acid is conveniently of a concentration of about 20%.The acidification drastically lowers the pH of the mixture to about 0-2,preferably about 0- 1, and thus enables a more rapid hydrolysis to thedesired hydroxylated isoflavone than would be achievable, albeit lesspracticably so, if the slightly acid (pH about 4-6) mixture were simplyheated without additional acidification. Following the addition of acidthe mixture is heated, optionally under increased pressure, to effectthe hydrolysis. In a preferred procedure, the solvent is continuallyremoved by distillation during the hydrolysis and continually replacedwith a lower alkanol, such as methanol or ethanol, or with water todilute the mixture; such solvent exchange is particularly practicablewhen a solvent with a lower boiling point than that of the added loweralkanol, e.g. acetone or tetrahydrofuran, has been employed previouslyas the reaction solvent. Otherwise), i.e. in those cases where a solventof about the same or a higher boiling point than that of the loweralkanol has been used, as much as possible of said solvent isconveniently first distilled off, optionally under reduced pressure, andthe lower alkanol added thereafter. In either case further acid may thenbe added to the mixture to to restore the pH value to about 0-2,preferably about 0-1. After the solvent exchange, and optionaladditional acidification, the mixture is conveniently heated, preferablyat reflux temperature, for a further period to complete the hydrolysis.In this case, too, it is convenient to observe the course of thehydrolysis (deacylation) by conventional analytical techniques such asHPLC in order to establish when the reaction has effectively beencompleted.

To isolate the desired product, directly from the mixture on completionof the reaction of the formic acid anhydride of the formula III with the2-hydroxydeoxybenzoin of the formula II or following the subsequentacid-catalysed hydrolysis for deacylation, this is convenientlycrystallized out by addition of water, suitably at elevated temperature,e.g. at about 50-60° C. The volume of added water is conveniently abouta fifth to about a half of the volume of the mixture containing theproduct before water addition. Optionally after also cooling the aqueouscrystalline medium, e.g. to a temperature from about room temperature toabout 0° C., the crystalline product is removed, e.g. by filtration, andif desired can be washed, e.g. with a mixture of ethanol and water, orotherwise purified by conventional methods, for example employing anorganic solvent in which the product is at the most sparingly soluble,and submitting the product to one or more recrystallizations. Especiallysuitable organic solvents for such purposes are ethanol, acetone,mixtures of both or mixtures of each with a relatively small proportionof water. The final step in such a purification procedure is usually athorough drying of the product at elevated temperature and reducedpressure.

The starting 2-hydroxydeoxybenzoins of the formula II are knowncompounds, obtainable by known procedures. For example, to obtain the2-hydroxydeoxybenzoin1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxy)-ethanone, i.e. the compound ofthe formula II wherein R¹ and R² each signify hydroxy,phloroglucinol(1,3,5-trihydroxybenzene) is reacted with4-hydroxyphenylacetonitrile in a Hoesch reaction [see Russ. Chem.Review. 31, 615-633 (1962)] to afford an intermediary, isolable iminiumsalt, which is then hydrolyzed to the desired product. Referencesconcerning this 2-hydroxydeoxybenzoin include W. Baker et al., J. Chem.Soc. 1926, 2713, and J. Chang et al., J. Agric. Food Chem. 42, 1869(1994). The further 2-hydroxydeoxybenzoins of formula II can be producedanalogously or are the subject of specific production processesdescribed in the scientific literature.

The further starting materials, i.e. the formic acid anhydrides of theformula III, are in some cases known. In general, such startingmaterials can be produced for example by reacting sodium formate withthe appropriate acid chloride of formula R³COCl in an organic solventsuch as an aliphatic or cyclic ether, e.g. diethyl ether ortetrahydrofuran, respectively; or an aromatic hydrocarbon, e.g. toluene[see, for example P. Strazzolini et al., Tetrahedron 46, 1081-1118(1990); R. Schijf et al., Rev. Trav. Chim. 85, 627 (1966); and L. I.Krimen, Org. Synth. 50, 1 (1970)]. A further known method for producingthe formic acid anhydride starting materials of the formula III involvesthe reaction of formic acid with the appropriate acid anhydride offormula (R³CO)₂O. Examples of such processes are published inter alia inR. Strazzolini et al. (as above); A. Béhal, Ann. Chim. 20, 411 (1900);W. Stevens et al., Rec. Trav. Chim. 83, 1287 (1964); and R. Schijf etal., Rec. Trav. Chim. 84, 594 (1965). A still further method,specifically for the production of those formic acid anhydrides of theformula III wherein R³ signifies carboxy-C₂₋₆-alkyl, a group—CH═CH—COOH, phenyl substituted with carboxyl, phenyl substituted withformyloxycarbonyl, a group —(CH₂)_(m)—COOH, a group —(CH₂)_(m)—COOCHO ora group —CH═CH—COOCHO (all these groups being ones terminating withcarboxyl or formyloxycarbonyl), involves the reaction of sodium formatewith the appropriate di(acid chloride), examples of such di(acidchloride) starting materials being maleic acid dichloride,1,2-dicarboxybenzene dichloride and succinic acid dichloride.

Some of the formic acid anhydrides of the formula III are known from thescientific literature, and these known examples, and some pertinentreferences in which they and (in most cases) their production aredescribed, are as follows:

-   -   R³=ethyl, n-propyl, isopropyl or tert. butyl: R. Schijf et al.,        Rev. Trans. Chim. 85, 627 (1966) and for ethyl and n-propyl,        additionally R. Schijf et al., ibid. 84, 594 (1965); and E. J.        Vlietstra et al., Recueil: J. of Royal Neth. Chem. Soc. 101,        460-462 (1982);    -   R³=n-butyl: E. J. Vlietstra et al. (as above);    -   R³=n-pentadecyl: Chem. Abs. 58469 (2001)    -   R³=styryl: W. K. Fife et al., J. Org. Chem. 51, 3746-3748        (1986);    -   R³=phenyl and 4-methoxyphenyl: W. K. Fife et al., as above; and        for phenyl additionally K. Kikukawa et al., J. Org. Chem. 46,        4413-4416 (1981) and G. F. Fanta, ibid. 29, 981 (1964);    -   R³=4-methylphenyl, 4-hexylphenyl and 4-phenylphenyl: W. K. Fife        et al., U.S. Pat. No. 4,874,558; and for 4-methylphenyl        additionally K. Kikukawa et al., as above;    -   R³=2-(methoxycarbonyl)-ethyl[—(CH₂)_(n)—COOR⁷ wherein n is 2 and        R⁷ is methyl]: F. Cavalli et al., Int. J. Chem. Kinet. 33,        431-439 (2001);    -   R³=ethoxy: T. Pavasavan, J. Org. Chem. 29, 3422-3423 (1964);    -   R³=formyloxy: G. Frapper, J.A.C.S. 122, 5367-1570 (2000);

Those starting materials, i.e. formic acid anhydrides, of the formulaIII, which are not previously known can be produced by analogous methodsto those for producing the known ones, i.e. analogous to the methodsdescribed above or which are indicated above by way of pertinentliterature references.

The invention is illustrated by the following Examples.

EXAMPLE 1

The apparatus consisted of a 250 ml double-walled reactor fitted with astirrer, a dropping funnel, a distillation column, argon gasificationmeans, a thermometer and a thermostat.

To a stirred suspension of 10 g (0.145 mol) of sodium formate in 25 g ofacetone at a temperature of 21-23° C. were added dropwise under an argonatmosphere 13 g (0.14 mol) of propionyl chloride. The mixture wasstirred at 22-25° C. for 2 hours, then warmed to 35° C., stirred for afurther hour and finally cooled to 20-23° C. Produced and present in themixture was the desired starting material propionyl formic anhydride.

Thereafter, a solution of 9.2 g (0.035 mol) of1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-ethanone and 3.6 g(0.035 mol) of triethylamine in 60 g of acetone was added dropwise tothe mixture containing propionyl formic anhydride, and the reactionmixture was stirred for 2 hours at 25° C. and for a further hour at 40°C., affording a beige-coloured suspension. To quench the reaction 10 gof ethanol were then added, the mixture was stirred for a further 30minutes, and the resulting suspension was allowed to stand at roomtemperature for about 16 hours.

To promote the hydrolysis 5 g of 50% sulphuric acid were added dropwiseto the suspension at room temperature. The mixture was heated to about60° C. to remove about 101 g of distillate. Following the addition of 68g of ethanol to replace the lost solvent a further 4 g of concentratedsulphuric acid were added dropwise, and the mixture was heated for afurther 90 minutes at about 70° C. The distillate which had accumulatedconsisted principally of acetone and a minor proportion of ethanol.

Following the distillation, 186 g of water were added to the remainingmixture in the reactor within 30 minutes, and the resulting slurry wasthen cooled to 10° C., stirred for a further 60 minutes and thenfiltered. The collected solid material was washed twice with 30 ml ofwater to afford 10.7 g of a moist) beige-coloured solid. This was driedat 100° C./1 mbar (0.1 kPa) for 2 hours to yield 8.4 g of an off-whitesolid consisting of genistein, of 98.9% purity according to HPLC. Theyield of genistein was calculated to be 88%.

6 g of the crude genistein product were dissolved in 180 g of ethanol atreflux temperature. Then 135 g of ethanol were distilled off underatmospheric pressure, and the resulting suspension was cooled to −20° C.and stirred for 1 hour at this temperature and subsequently filtered.The collected solid material was washed with 10 g of cold ethanol, andthen dried for 2 hours at 100° C./1 mbar (0.1 kPa) to give 5 g of awhite solid. The genistein had been produced in a purity of 99.7%(according to HPLC). The yield after this purification was 84%.

Analytical and spectral data of the product:

¹H-NMR (400MHz, d₆-DMSO): 13.0 (s, OH), 10.9 (s, OH), 9.6 (s, OH), 8.33(s, C(2)-H), 7.37 (d, J=8 Hz, C(2′)H and C(6′)H), 6.81 (d, J=8 Hz,C(3′)H and C(5′)H), 6.38 (d, J˜2 Hz, 1 H), 6.22 (d, J ˜2 Hz, 1 H);

Mass spectroscopy: 270.2 (M⁺, 100%);

Infrared (Nujol, cm⁻¹): 3411 (OH), 1654 (C═O);

Ultraviolet (ethanol): 261 nm (ε=73570, log ε=4.87);

Content acc. to HPLC: 99.7% (as given above);

M. Pt.: 303° C.

EXAMPLE 2

The apparatus was essentially the same as that described in Example 1,the reactor having a capacity of 500 ml, however.

46.3 g (0.5 mol) of propionyl chloride were added dropwise to a stirredsuspension of 35.7 g (0.52 mol) of sodium formate and 26.3 g (0.1 mol)of 1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-ethanone in 180 g ofacetone at a temperature of 21-23° C. under an argon atmosphere. Themixture was stirred at 23-25° C. for 1 hour, and then heated for 1 hourat 35° C. To the suspension at 18-20° C. were added 10.1 g (0.1 mol)triethylamine. The reaction mixture was stirred at 21-22° C. for 2 hoursand then heated 1 hour at 35° C. To quench the reaction 50 g of ethanolwere then added and the mixture was stirred for a further 15 minutes.

To promote the hydrolysis 20 g of 50% sulphuric acid were added dropwiseto the suspension, which was then heated to about 60° C. to remove 215 gof distillate. The lost solvent was replaced with 80 g of ethanol. Thena further 15 g of concentrated sulphuric acid were added dropwise, andthe mixture heated for a further 90 minutes at about 70° C.

350 g of water were added to the mixture over 30 minutes. The resultingslurry was cooled, held 1 hour at 10° C., and filtered. The collectedsolid was washed twice with 40 g water and then with 40 g of 50% aqueousethanol to afford 31 g of a moist beige-coloured product. This was thendried at 100° C./1 mbar (0.1 kPa) for 2 hours to afford 25.3 g of anoff-white product, which according to HPLC analysis consisted of 98.4%by weight of genistein. The calculated yield of genistein was 92.2%.

20 g of the crude genistein product were suspended in 120 g of 35%aqueous ethanol, and the suspension was stirred at reflux temperaturefor 2 hours. The resulting slurry was cooled down to 0° C., stirred for1 hour at that temperature and filtered. The collected solid was washedwith 20 g of 35% aqueous ethanol and then dried at 100° C./1 mbar (0.1kPa) for 2 hours to afford 19.5 g of an off-white product. HPLC analysisindicated that this consisted of 99% by weight of genistein. The yieldof genistein after this further purification was 98%.

EXAMPLE 3

The apparatus was essentially the same as that described in Example 2.

55.5 g (0.6 mol) propionyl chloride were added dropwise at a temperatureof 21-23° C. to a stirred suspension of 42.1 g (0.62 mol) of sodiumformate in 90 g acetone under an argon atmosphere. The mixture wasstirred at 25° C. for 2 hours, then heated for 1 hour at 35° C. Asolution of 26.3 g (0.1 mol) of1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-ethanone and 20.4 g (0.2mol) triethylamine in 165 g of acetone was added dropwise to theresulting suspension at 20-22° C. The reaction mixture was stirred at 21-22° C. for 2 hours. To quench the reaction 50 g of ethanol were thenadded and the mixture was stirred for a further 30 minutes.

To promote the hydrolysis 20 g of 37% aqueous hydrochloric acid wereadded dropwise and the mixture was allowed stand at room temperature forabout 16 hours. The mixture was then heated to about 60° C. to remove303 g of distillate. The lost solvent was replaced with 100 g ofethanol. Then 20 g of 37% aqueous hydrochloric acid were added dropwise,and the mixture heated for a further 90 minutes at about 70° C. 400 g ofwater were added to the resulting suspension at 75-80° C. over 30minutes. The slurry was cooled, held 1 hour at 10° C., and thenfiltered. The solid was washed twice with 50 g water to afford 31.5 g ofa moist beige-coloured product. This was then dried at 100° C./1 mbar(0.1 kPa) for 2 hours to afford 25.5 g of an off-white product. HPLCanalysis indicated that this consisted of 98.3% by weight of genistein.The calculated yield of genistein was 93%.

20 g of the crude genistein product were dissolved in 600 g of ethanolat reflux temperature. Then 470 g of ethanol were distilled off underatmospheric pressure and 130 g of water were added dropwise at 75-80° C.The resulting suspension was cooled to 0° C. and stirred one hour atthis temperature, then filtered. The filter cake was washed with 20 g of50% aqueous ethanol, then dried at 100° C./1 mbar (0.1 kPa) for 2 hoursto afford 19 g of a white product. HPLC analysis indicated that thisconsisted of 99.1% by weight of genistein. The yield of genistein afterthis further purification was 95.7%.

EXAMPLE 4

The apparatus was the same as that described in Example 1.

27.8 g (0.3 mol) of propionyl chloride were added dropwise to asuspension of 21.1 g (0.31 mol) of sodium formate and 13.1 g (0.05 mol)of 1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-ethanone in 100 g ofethyl formate at a temperature of 23-26° C. under argon. The suspensionwas stirred for 2 hours at 25° C. and heated for 1 hour at 35° C. 10.2 g(0.1 mol) of triethylamine were added to the resulting mixture at aninternal temperature of 18-22° C. The mixture was then stirred for 16hours at room temperature. Then 20 g of ethanol were added and thesuspension was stirred for 15 minutes.

Thereafter, to promote the hydrolysis, 20 g of 50% sulphuric acid wereadded dropwise and the mixture was heated to about 80° C. to remove 128g of distillate. The lost solvent was replaced with 50 g of ethanol. 150g of water were added over 30 minutes to the mixture. The resultingslurry was cooled, held for 1 hour at 10° C., and then filtered. Thesolid was washed twice with 25 g water and then with 30 g of 50% aqueousethanol to afford 13.6 g of a moist beige-coloured product. This wasthen dried at 100° C./1 mbar (0.1 kPa) for 2 hours to afford 12.7 g of abeige-coloured product. HPLC analysis indicated that this consisted of98.7% by weight of genistein. The yield of genistein was 92.7%.

EXAMPLE 5

The apparatus was the same as that described in Example 1.

27.8 g (0.3 mol) of propionyl chloride were added dropwise to 21..1 g(0.31 mol) of sodium formate at a temperature of 23-25° C. under argon.The white suspension was stirred 2 hours at 25° C. and heated for 1 hourat 35° C. To this mixture were added 13.1 g (0.05 mol) of1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-ethanone and 10.2 g (0.1mol) of triethylamine at an internal temperature of 20-22° C. Themixture was then stirred at 21-22° C. for 2 hours and heated for 2 hoursat 35° C. 50 g of ethanol were added at 20° C. and the suspension wasstirred for 15 minutes.

Thereafter, to promote the hydrolysis, 30 g of 50% sulphuric acid wereadded dropwise and the mixture was heated for 1 hour at 75° C. To themixture were then added over 30 minutes 150 g of water. The resultingslurry was cooled, held for 1 hour at 10° C., and then filtered. Thesolid was washed twice with 20 g water then with 30 g of 50% aqueousethanol to afford 13.6 g of a moist beige-coloured product. This wasthen dried at 100° C./1 mbar (0.1 kPa) for 2 hours to give 10.9 g of abeige-coloured product. HPLC analysis indicated that this consisted of98.4% by weight of genistein. The yield of genistein was 79.5%.

EXAMPLE 6

The apparatus was the same as that described in Example 1.

A suspension of 21.1 g (0.31 mol) of sodium formate, 13.1 g (0.05 mol)of 1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-ethanone and 6.9 g(0.05 mol) potassium carbonate in 100 g of acetone was stirred 1 hour at25° C. under argon. To this mixture were added dropwise 27.8 g (0.3 mol)of propionyl chloride at a temperature of 21-23° C. The suspension wasstirred for 16 hours at room temperature. Then 20 g of ethanol wereadded, and the suspension was stirred for 15 minutes.

Thereafter, to promote the hydrolysis, 20 g of 50% sulphuric acid wereadded dropwise and the mixture was heated to about 60° C. to remove thesolvent. The lost solvent was replaced with 50 g of ethanol. Then afurther 10 g of concentrated sulphuric acid were added dropwise, and themixture heated for a further hour at about 80° C. To the mixture wasadded over 30 minutes 120 g of water. The slurry was cooled, held for 1hour at 10° C. then filtered. The solid was washed twice with 20 g waterand then with 30 g of 50% aqueous ethanol. This was then dried at 100°C./1 mbar (0.1 kPa) for 2 hours to give 9.3 g of a pale yellow product.HPLC analysis indicated that this consisted of 97.8% by weight ofgenistein. The yield of genistein was 67.5%.

EXAMPLE 7

The apparatus was the same as that described in Example 1.

27.8 g (0.3 mol) of propionyl chloride were added dropwise to a stirredsuspension of 21.1 g (0.31 mol) of sodium formate and 13.1 g (0.05 mol)of 1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-ethanone in 100 g ofdimethylformamide at a temperature of 25-28° C. under an argonatmosphere. The mixture was stirred at 25° C. for 2 hours and thenheated for 2 hours at 35° C. To quench the reaction 15 g of ethanol werethen added and the mixture was stirred for a further 15 minutes.

To promote the hydrolysis, 20 g of 50% sulphuric acid were addeddropwise to the suspension at room temperature, and the reaction mixturewas distilled in vacuo at 80 mbar (8 kPa) and 70-80° C. to remove 75 gof solvent. Then a further 20 g of concentrated sulphuric acid wereadded dropwise, and the mixture was heated for a further hour at 80° C.To the mixture was added 150 g of water, which promoted crystallization.The resulting slurry was cooled, held for an hour at 10° C., and thenfiltered. The collected solid was washed twice with 25 g of water andonce with 30 g of 50% aqueous ethanol. The solid was then dried at 10°C./1 mbar (0.1 kPa) for 2 hours to afford 10.5 g of an off-whiteproduct. HPLC analysis indicated that this consisted of 98.2% by weightof genistein. The yield of genistein was 77%.

EXAMPLE 8

The apparatus was the same as that described in Example 1.

11.8 g (0.255 mol) of formic acid were added dropwise under argon to32.6 g (0.25 mol) of propionic acid anhydride at an internal temperatureof 25° C. The mixture was stirred at 45° C. for 2 hours, then cooled to20° C. A solution of 13.1 g (0.05 mol) of1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-ethanone in 70 g ofacetone was then added at 20-22° C., followed by 5.1 g (0.05 mol) oftriethylamine. The mixture was stirred at 22-23° C. for 2 hours. Afterthe addition of a further 5.1 g of triethylamine, the mixture wasstirred at room temperature for about 16 hours and then after warming at40° C. for a further hour at this temperature. To quench the reaction,25 g of ethanol were added and the mixture was stirred for 15 minutes.

Thereafter, to promote the hydrolysis, 10 g of 50% sulphuric acid wereadded dropwise and the mixture was heated to 60° C. to remove 85 g ofdistillate. The lost solvent was replaced with 50 g of ethanol. Then 10g of concentrated sulphuric acid were added dropwise, and the mixtureheated for a further 90 minutes at about 70° C. 180 g of water wereadded to the mixture over 30 minutes. The resulting slurry was cooled,held for 1 hour at 10° C., and then filtered. The solid was washed twicewith 25 g of water then with 20 g of 50% aqueous ethanol to afford 14.2g of a moist beige-coloured product. This was then dried at 100° C./1mbar (0.1 kPa) for 2 hours to afford 11.3 g of an off-white product.HPLC analysis indicated that this consisted of 99% by weight ofgenistein. The yield of genistein was 86.5%.

EXAMPLE 9

The apparatus was the same as that described in Example 1.

11.8 g (0.255 mol) of formic acid were added dropwise under argon to32.6 g (0.25 mol) of propionic acid anhydride and 13.1 g (0.05 mol) of1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-ethanone at an internaltemperature of 25° C. The mixture was then stirred at 45° C. for 2 hoursand cooled to 20° C. Then 5.1 g of triethylamine (0.05 mol) were addeddropwise, and the mixture was stirred at 22° C. for 2 hours, after whicha final 5.1 g of triethylamine were added and the mixture was stirred atroom temperature for about 16 hours. The mixture was warmed to 40° C.and stirring continued for a further hour at this temperature. To quenchthe reaction, 25 g of ethanol were added and the mixture was stirred for15 minutes.

Thereafter, to promote the hydrolysis, 10 g of 50% sulphuric acid wereadded dropwise and the mixture was heated for 1 hour at 75° C. Then 10 gof concentrated sulphuric acid and 35 g of ethanol were added dropwise,and the stirring continued for a further 90 minutes at this temperature.180 g of water were added over 30 minutes to the mixture. The resultingslurry was cooled, held for 1 hour at 10° C., and then filtered. Thesolid was washed twice with 25 g of water then with 20 g of 50% aqueousethanol to afford 13.6 g of a moist beige-coloured product. This wasthen dried at 100° C./1 mbar (0.1 kPa) for 2 hours to afford 11.3 g ofan off-white product. HPLC analysis indicated that this consisted of98.5% by weight of genistein. The yield of genistein was 82.5%.

EXAMPLE 10

The apparatus was essentially the same as that described in Example 1.

42.1 g (0.3 mol) benzoyl chloride were added dropwise under an argonatmosphere to a stirred suspension of 21.1 g (0.31 mol) of sodiumformate in 45 g of acetone at a temperature of 21-23° C. The mixture wasstirred at 25° C. for 2 hours, then heated for 1 hour at 40° C. To theresulting suspension at 20-22° C. was added dropwise a solution of 13.1g (0.05 mol) 1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-ethanoneand 10.2 g (0.1 mol) triethylamine in 75 g of acetone. The reactionmixture was stirred at 21-22° C. for 2 hours, then heated for 1 hour at35° C. Then 20 g of ethanol were added, and the mixture was stirred for15 minutes.

To promote the hydrolysis 20 g of 50% aqueous sulphuric acid were addeddropwise and the mixture was allowed stand at room temperature for about16 hours. The mixture was then heated to about 60° C. to remove thesolvent. The lost solvent was replaced with 50 g of ethanol. Then 15 gof concentrated sulphuric acid were added dropwise, and the mixture washeated for a further 3.5 hours at about 70° C. 100 g of water were addedto the resulting suspension at 75-80° C. over 30 minutes. The mixturewas cooled at 20° C. and extracted four times with 100 ml of ethylacetate. The organic layer was then heated to 40° C./150 mbar (15 kPa)to remove about 300 ml of distillate. The slurry was cooled, allowedstand at room temperature for about 16 hours and filtered. The solid waswashed with 10 ml of ethyl acetate to afford 11.5 g of a moistbeige-coloured product.

The crude genistein product was suspended in 60 g of 50% aqueousethanol, and the suspension was stirred at reflux temperature for 1hour. The resulting suspension was cooled to 5° C. and stirred for onehour at this temperature, then filtered. The filter cake was washed with10 g of 50% aqueous ethanol, then dried at 100° C./1 mbar (0.1 kPa) for2 hours to afford 7.3 g of an off-white product. HPLC analysis indicatedthat this consisted of 97.5% by weight of genistein. The yield ofgenistein after this further purification was 52.7%. 1

EXAMPLE 11

The apparatus was the same as that described in Example 2.

55.4 g (0.5 mol) of ethyl chloroformate were added dropwise under argonto a suspension of 35.7 g (0.51 mol) of sodium formate and 26.3 g (0.1mol) of 1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-ethanone in 150g of acetone at a temperature of 0-5° C. The suspension was stirred for3 hours at 0-5° C. To the resulting mixture were added dropwise 15.2 g(0.15 mol) of triethylamine at an internal temperature of 0-5° C. Themixture was then stirred for 1 hour at 0-5° C. and for 16 hours at roomtemperature.

The mixture was heated to about 60° C. to remove 160 g of distillate.The lost solvent was replaced with 80 g of ethanol. Thereafter, 50 g of50% sulphuric acid were added over 15 minutes and the mixture was heatedfor 3 hours at about 70° C. To the mixture were added over 30 minutes350 g of water. The resulting slurry was cooled, held for 1 hour at 30°C., and then filtered. The solid was washed twice with 40 g of water andonce with 50 g of 50% aqueous ethanol to afford 32.1 g of a moistbeige-coloured product. This was then dried at 100° C./1 mbar (0.1 kPa)for 2 hours to afford 26.3 g of a beige-coloured product. HPLC analysisindicated that this consisted of 81.1% by weight of genistein.

1 g of the crude genistein product were dissolved in 25 g of ethanol atreflux temperature. Then 5 g of 50% sulphuric acid were added dropwiseand the mixture heated for a further 2 hours to about 100° C. under apressure of 2 bar (0.2 MPa). Then 15 g of ethanol were distilled offunder atmospheric pressure, and 5 g of water were added dropwise at75-80° C. The resulting suspension was cooled to 20° C. and stirred 2hours at this temperature, then filtered. The collected solid was driedat 100° C./1 mbar (0.1 kPa). for 2 hours to afford 0.9 g of an off-whiteproduct. HPLC analysis indicated that this consisted of 98.5% by weightof genistein.

EXAMPLE 12

The apparatus was the same as that described in Example 2.

60 g (0.65 mol) of propionyl chloride were added dropwise to a stirredsuspension of 51 g (0.75 mol) of powdered sodium formate and 26.3 g (0.1mol) of 1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-ethanone in 200g of acetone at a temperature of 21-23° C. The suspension was stirred at23-25° C. for 30 minutes then heated for 13 hours at 32° C.

Thereafter, to promote the hydrolysis, 50 g of 50% sulphuric acid wereadded dropwise at 10-12° C. and the mixture was heated to about 60° C.to remove 204 g of distillate. The lost solvent was replaced with 120 gof ethanol. Then a further 25 g of 50% sulphuric acid were addeddropwise, and the mixture heated for a further 1 hour at about 72° C. Tothe mixture was added over 30 minutes 300 g of water. The slurry wascooled, held for 1 hour at 30° C. and then filtered. The solid waswashed twice with 40 g of water and once with 50 g of 50% aqueousethanol. This was then dried at 100° C./1 mbar (0.1 kPa) for 2 hours toafford 21.6 g of a white product. HPLC analysis indicated that thisconsisted of 99.3% by weight of genistein. The yield of genistein was79.4%.

EXAMPLE 13

The apparatus was the same as that described in Example 2.

54.4 g (0.5 mol) of isobutyryl chloride were added dropwise under argonto a suspension of 35.7 g (0.51 mol) of sodium formate and 26.3 g (0.1mol) of1-(2,4,6-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-ethanone in 150 gof acetone at a temperature of 21-23° C. The suspension was stirred for1 hour at 23-25° C. and heated for 2 hours at 30-32° C. To the resultingmixture were added 15.2 g (0.15 mol) of triethylamine at an internaltemperature of 18-22° C. The mixture was then stirred for 1 hour at21-22° C. and heated at 30-32° C. for 1 hour.

Thereafter, to promote the hydrolysis, 50 g of 50% sulphuric acid wereadded dropwise and the mixture was heated to about 60° C. to remove 148g of distillate. The lost solvent was replaced with 120 g of ethanol.The mixture was heated for a further 3 hours at about 70° C. To thesuspension were added over 30 minutes 350 g of water. The resultingslurry was cooled, held for 1 hour at 30° C., and then filtered. Thesolid was washed twice with 40 g water and once with 50 g of 50% aqueousethanol to afford 31.6 g of a moist off-white product. This was thendried at 100° C./1 mbar (0.1 kPa) for 2 hours to afford 24.5 g of anoff-white product. HPLC analysis indicated that this consisted of 99.6%by weight of genistein. The yield of genistein was 90.3%.

1. A process for manufacturing a hydroxylated isoflavone of the generalformula

wherein R¹ signifies hydrogen or hydroxy, and R² signifies hydroxy or C₁_(—) ₆-alkoxy, comprising reacting a 2-hydroxydeoxybenzoin of thegeneral formula

wherein R¹ and R² are defined as above, with a formic acid anhydride ofthe general formula

wherein R³ signifies C₂₋₂₀-alkyl, C₁₋₆-haloalkyl, (C₁₋₆-alkoxy)methyl,carboxy-C₂₋₆-alkyl, aryl-C-₁₋₆-alkyl, a group —CH₂—(OCH₂CH₂)_(m)—O—C₁_(—) ₆-alkyl, a group —CH(R⁴)═CR⁵R⁶, a group —CH═CH—COOH,C₃₋₈-cycloalkyl, aryl, heteroaryl, di(C₁ _(—) ₆-alkyl)aminomethyl,diarylaminomethyl, a group —(CH₂)_(n)—COOR⁷, a group —(CH₂)_(m)—COOCHO,a group —CH═CH—COOCHO, C₁₋₆-alkoxy, aryloxy or formyloxy, each of R⁴, R⁵and R⁶, independently, signifies hydrogen, C₁₋₆-alkyl or aryl, R⁷signifies hydrogen, C₁₋₆-alkyl or aryl, m signifies an integer 1 to 4,and n signifies zero or an integer 1 to 8, in the presence of a base orin a solvent which acts as a base, and if necessary promoting theensuing hydrolysis of the so-produced acylated form of the hydroxylatedisoflavone of the formula I by acidification.
 2. The process accordingto claim 1, wherein a base is employed and the process is carried outadditionally in an organic solvent.
 3. The process according to claim 2,wherein the base is an alkali metal or alkaline earth metal hydroxide,an alkali metal or alkaline earth metal carbonate or bicarbonate, analkali metal or alkaline earth metal salt of a carboxylic acid with upto 10 carbon atoms, an aliphatic or mixed aliphatic/aromatic tertiaryamine, a nitrogen-containing heterocyclic base, or a secondary ortertiary phosphate.
 4. The process according to claim 3, wherein thebase is lithium, sodium or potassium hydroxide, lithium, sodium orpotassium carbonate, calcium or magnesium carbonate, lithium, sodium orpotassium bicarbonate, calcium or magnesium bicarbonate, sodium formate,potassium propionate, trimethylamine, triethylamine,N-ethyldiisopropylamine, N,N-dimethylethanolamine,2-(dimethylamino)-ethyl acetate, triethanolamine, N,N-dialkylaniline,optionally alkyl substituted pyridine, a N-alkyl substituted piperidine,a N-alkyl substituted morpholine, imidazole, trisodium phosphate ortripotassium phosphate.
 5. The process according to claim 3, wherein thebase is an alkali metal carbonate, bicarbonate or formate, or analiphatic or mixed aliphatic/aromatic tertiary amine.
 6. The processaccording to claim 2, wherein the solvent is an aliphatic or cyclicether, a lower aliphatic nitrile, a lower aliphatic ester,dimethyl-sulphoxide, a halogenated, particularly chlorinated, loweraliphatic hydrocarbon, an aromatic hydrocarbon or a lower aliphaticketone.
 7. The process according to claim 6, wherein the solvent isdiethyl ether, diisopropyl ether, dibutyl ether, tert. butyl methylether, diethylene glycol dimethyl ether, tetrahydrofuran, dioxan,acetonitrile, a lower alkyl formate or acetate, dimethylsulphoxide,methylene chloride, benzene, toluene, an xylene, acetone, 2-butanone,diethyl ketone or methyl isobutyl ketone.
 8. The process according toclaim 1, wherein the process is carried out in a solvent which acts as abase, and said solvent is a di(lower alkyl)formamide, preferablydimethylformamide, dimethylacetamide, tetramethylurea orN-methyl-pyrrolidone.
 9. The process according to claim 1, wherein theprocess is carried out at temperatures in the range of about −20° C. toabout +80° C., preferably at temperatures from about −5° C. to about+45° C.
 10. The process according to claim 1, wherein about 1.5 to about6 equivalents, preferably about 2.5 to about 5 equivalents, of theformic acid anhydride of the formula III are present in the reactionmixture per equivalent of the 2-hydroxydeoxy-benzoin of the formula II.11. The process according to claim 2, wherein the base is present in thereaction mixture in an amount which is up to about 6 equivalents perequivalent of the 2-hydroxydeoxybenzoin of the formula II.
 12. Theprocess according to claim 1, wherein the hydrolysis of any producedacylated form of the desired hydroxylated isoflavone of the formula I inthe mixture after reaction is effected by addition of aqueous mineralacid to lower the pH of the mixture to about 0-2, preferably about 0-1,and heating the acidified mixture.
 13. The process according to claim12, wherein the solvent is continually removed by distillation duringthe hydrolysis and continually replaced with a lower alkanol, preferablymethanol or ethanol, or with water to dilute the mixture, or wherein asmuch as possible of the solvent is first distilled off, optionally underreduced pressure, and the lower alkanol or water added thereafter, andin either case further acid is then optionally added to the mixture torestore the pH value to about 1-2, preferably about 0-1, and the mixtureis heated, preferably at reflux temperature, for a further period tocomplete the hydrolysis.
 14. The process according to claim 1, whereinthe desired product of the formula I is isolated, either directly fromthe mixture on completion of the reaction of the formic acid anhydrideof the formula III with the 2-hydroxydeoxybenzoin of the formula II orfollowing the subsequent acid-catalysed hydrolysis for deacylation, bycrystallization induced by addition of water and removal of theresulting crystalline product by filtration.
 15. The process accordingto claim 1, wherein propionic formic anhydride is used as the formicacid anhydride of the formula III.
 16. The process according to claim 1,wherein isobutyric formic anhydride, carbonic monoformic anhydridemethyl ester or carbonic monoformic anhydride ethyl ester is used as theformic acid anhydride of the formula III.